ROLE OF THE F-BOX PROTEIN POF6 IN CELL CYCLE

F-BOX 蛋白 POF6 在细胞周期中的作用

• 批准号: 7957672
• 负责人: TAKASHI TODA
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957672
• 关键词: Adaptor Signaling Protein; Affect; Binding; Biogenesis; Biology; Cell Cycle; Cell Cycle Progression; Cell Size; Cell Survival; Cells; Centrifugation; Characteristics; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA biosynthesis; Data; Defect; F Box Domain; F-Box Proteins; Fission Yeast; Functional disorder; Funding; Fungal Genome; Genes; Grant; Growth; Homologous Gene; Institution; Investigation; Lead; Mass Spectrum Analysis; Molecular Weight; Mono-S; Peptides; Phenotype; Ploidies; Polyribosomes; Proteins; Recycling; Research; Research Personnel; Resources; Ribosomes; Role; Saccharomyces cerevisiae; Sampling; Scaffolding Protein; Silver Staining; Source; Sucrose; Terminator Codon; Translating; Translation Initiation; Translations; United States National Institutes of Health; cell growth; inhibitor/antagonist; mutant; protein function

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The S. pombe F-box protein Pof6 is essential for cell viability and contains an F-box in its amino-terminal portion, two Sec10 domains in the carboxy-terminal portion and a CAAX domain prior to the stop codon. Its closest homologue is Rcy1 from S. cerevisiae (24% identity), a non-essential F-box protein involved in protein recycling (which also contains a Sec10 domain and a CAAX domain). Pof6 has been shown to interact with Skp1 (the F-box-binding adaptor protein) consistently and, most of the residues important for the Skp1/F-box domain interface are conserved in the F-box of Pof6. Surprisingly, unlike other F-box proteins, Pof6 was found not to interact with the scaffold protein Cul1 raising the possibility that this F-box protein is part of a non-SCF complex. This hypothesis is supported by the fact that the residues important for the Cul1/F-box domain interface are not conserved in Pof6. To decipher the essential role for Pof6 in the cell, a TAP purification followed by MudPIT analysis was performed in collaboration with Dr. J. Thompson and Dr J. Yates, TSRI, San Diego. Among several new Pof6 interactors, a protein of high molecular weight, visible on purified silver-stained samples and well detected by mass spectrometry (139 unique peptides/792 total peptides) was identified. The gene encoding for this protein is essential in S. pombe and its interaction with Pof6 has been confirmed by coimmunoprecipitation. The function of this protein is now under investigation. The phenotypic characterization of the ts pof6 mutants, pof6-1166 and pof6-51 reveals similar defects for both strains: small sized cells with septation defects and an accumulation of cells containing 4C DNA content. These phenotypes suggest that DNA replication is not affected and that mutant cells are defective in cell growth rather than cell cycle progression. Altogether, the data collected so far lead us to the hypothesis that Pof6 function is related to the translation machinery as it seems to consistently interact with ribosome components, therefore its dysfunction would result in growth defects. As a result both pof6 ts strains appear to be sensitive to translation inhibitors, supporting the idea that Pof6 directly modulates the biogenesis or the activity of the ribosomes. Subsequently polysome profiling analysis (sucrose gradient centrifugation) showed that Pof6 co-sediments with the active polysomes, the 80S and 60S complexes. A portion of Skp1 co-migrates with Pof6, although most of the protein remains with smaller molecular weight complexes (i.e. SCF complexes). Inactivation of Pof6 modifies dramatically the polysome profile: the translating polysomes regressed and seem to accumulate into inactive mono-ribosomes, resulting in the increase of the 80S peak. This figure is characteristic of a diminution in translation activity. We are now planning to use the polysome profiling analysis to examine whether the Pof6 interactor co-sediments with Pof6 and Skp1 at the high molecular weight complexes, and investigate if Pof6 is involved in translation initiation and/or elongation.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 S. pombe F-box蛋白POF6对于细胞活力是必不可少的，并在其氨基末端部分中包含一个F-box，羧基末端部分中的两个SEC10域和一个在终止密码子之前的CAAX域。它最接近的同源物是酿酒酵母的RCY1（24％身份），这是一种参与蛋白质回收的非必需的F-box蛋白（还包含SEC10结构域和CAAX结构域）。 POF6已显示与SKP1（F-box结合衔接蛋白）相互作用，并且对于SKP1/F-Box域界面而言，大多数残基在POF6的F-box中保守。令人惊讶的是，与其他F-box蛋白不同，POF6被发现不与支架蛋白CUL1相互作用，从而增加了该F-box蛋白是非SCF复合物的一部分的可能性。这一假设得到了以下事实，即对CUL1/F-Box域界面重要的残基在POF6中不保守。 为了破译POF6在细胞中的重要作用，与J. Thompson博士和圣地亚哥TSRI的J. Thompson博士合作进行了TAP纯化，然后进行了Mudpit分析。在几个新的POF6相互作用器中，一种高分子量的蛋白质，可在纯化的银染色样品上可见，并通过质谱法（139个独特的肽/792个总肽）可见。该蛋白质编码的基因在S. pombe中至关重要，并且通过共免疫沉淀证实了其与POF6的相互作用。该蛋白的功能现在正在研究中。 TS POF6突变体，POF6-1166和POF6-51的表型表征揭示了这两种菌株的相似缺陷：具有分离缺陷的小细胞和含有4C DNA含量的细胞积累。这些表型表明DNA复制不受影响，突变细胞在细胞生长而不是细胞周期进程中有缺陷。总体而言，迄今为止收集的数据使我们提出了这样的假设：POF6函数与翻译机制有关，因为它似乎与核糖体成分持续相互作用，因此其功能障碍会导致生长缺陷。结果，两种POF6 TS菌株似乎对翻译抑制剂敏感，支持POF6直接调节核糖体的生物发生或活性的想法。 随后，多元分析分析（蔗糖梯度离心）表明，POF6与活性多聚体，80s和60s复合物的同型。尽管大多数蛋白质保留为较小的分子量复合物（即SCF复合物），但SKP1的一部分与POF6共移民。 POF6的灭活会显着修饰多聚合体的轮廓：翻译多粒子体回归并似乎积聚成非活动的单核体，从而导致80年代峰的增加。这个数字是翻译活动减少的特征。现在，我们计划使用多多元体分析分析来检查高分子量复合物与POF6和SKP1的POF6相互作用的共同符号，并研究POF6是否参与翻译启动和/或延长。